Assay apparatus and methods

ABSTRACT

Apparatus and methods for determining whether a test compound induces cell activity, changes cell activity, prevents cell activity, or inhibits cell activity. An embodiment comprises placing a test compound solution in contact with a cell suspension media containing cells, diffusing the test compound solution into the cell suspension from one or more sides, and detecting activity in the cells with respect to their distance from the side from which the test compound is diffusing. Embodiments may provide an apparatus that allows a side source, a point source, or both, from which a test compound solution diffuses into a cell suspension media and contacts cells. Detecting cell activity may involve detecting activity in a first cell group proximate to the side from which the test compound is diffusing, and detecting activity in a second cell group farther than the first cell group from the side from which the test compound is diffusing.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/213,632,filed Jul. 19, 2016, now U.S. Pat. No. 10,384,207, issued Aug. 20, 2019,which claims the benefit of U.S. Provisional Application No. 62/194,888,filed Jul. 21, 2015, both of which are herein incorporated by referencein their entirety.

BACKGROUND Field

The present embodiments relate to methods and apparatus for high contentscreening (HCS) and high throughput screening (HTS) and basic (lowthroughput) research. In particular, the present embodiments relate tocell activity assays (CAA) that prevent cell activity or inhibit cellactivity, or involve chemotaxis, migration, angiogenesis, growth,proliferation, and other cell activity based on, for example,morphology, shape, and movement of cells. The present embodiments alsorelate to cell activity assays involving changes internal to cells suchas differentiation, alteration of metabolic rate, and movement ofmolecules within a cell initiated by activation of receptors in the cellmembrane. The present embodiments also relate to cell activity assaysinvolving the interaction of cells in response to various chemicalenvironments, and the interaction of different cell types with oneanother. The present embodiments also relate to assays involving thepenetration of cell layers by chemical compounds or other entities, andto assays for ascertaining the diffusion rate of members of a compoundlibrary through various confluent cell layers, e.g., endothelial orepithelial cell layers.

Background

Pharmaceutical companies expend considerable resources on researchingand developing drug therapies. The research and development process,from conception to eventual approval by the Food and Drug Administration(FDA), can last several years. Thus, in the initial stages, it is highlydesirable to quickly rule out unusable chemical substances and focusefforts on effective substances. In particular, there is a need forrapidly and accurately assessing whether and to what extent cellactivity is affected by compounds.

In addition, pharmaceutical companies and research institutions maydesire flexible assay platforms that can be conveniently customized forparticular studies or applications. Budget-conscious researchers mayappreciate assay platforms that can be customized and assembled for afirst study, and then disassembled, sterilized, and customized andre-assembled for a second different study.

SUMMARY

Embodiments provide apparatus and methods for rapidly and accuratelyassessing whether and to what extent cell activity is affected bycompounds that make contact with a living cell's membrane. The presentembodiments may perform this assessment with minimal cost in cells,reagents, assay platforms, and other disposables, and with very lowcoefficients of variability, thus allowing primary screening of largecompound libraries without using duplicate or triplicate sites for eachcompound. The present embodiments may provide an assay apparatus forHCS, HTS, CBHTS, and cell based screening, as well as basic research incell activity. The apparatus and methods of the present embodimentsfacilitate cell activity assays. In particular, the present embodimentsmay provide means for assessing whether compounds from a compoundlibrary can induce cell activity, e.g., chemotaxis. In contrast to theprior art, the present embodiments may allow for the determination ofcell activity by detecting changes in cells that occur well before acell could migrate through a membrane. These changes may include, forexample, cell orientation, internal morphological changes, temperaturevariations, molecular movement within the cell, and electromagneticchanges—in short, any change in cells that can be detected.

Pharmaceutical companies expend considerable resources on researchingand developing drug therapies. The research and development process,from conception to eventual approval by the Food and Drug Administration(FDA), can last several years. Thus, in the initial stages, it is highlydesirable to quickly rule out unusable chemical substances and focusefforts on effective substances. In developing drug therapies,pharmaceutical companies typically start with a vast library ofchemicals. From this library, a large number of the chemicals may havethe potential to therapeutically act on the cells associated with thedisease or ailment for which the drug is being developed. Determiningwhich chemicals affect the cells is therefore an important step in drugdevelopment.

Chemotaxis is the directional movement (migration) of biological cellsor organisms in response to concentration gradients of chemicals.Invasion is the movement (migration) of cells into or through a barrier.Tumor invasion is such action initiated by cancer cells into or throughbiological tissue in vivo, or into or through extra cellular matrixproteins, e.g., collagen or matrigel, or into or through barriers madeof other substances, in vitro. Angiogenesis is the migration andformation of capillary blood vessels by endothelial cells. Growth is theincrease in the size, form, or complexity of cells. Proliferation is thegrowth of cells by cell division. Differentiation is the process bywhich cells change from a less specialized to a more specialized stateusually associated with different functional roles and the expression ofnew and different traits.

Interaction of cells is the alteration of cell behavior such asmovement, invasion, angiogenesis, growth, proliferation, ordifferentiation in response to the presence and action of nearby cellsof the same or different type. The movement of compounds and structureswithin cells is another kind of cell activity that can be of interest indrug discovery. For example, powerful optical detection systems maytrack the movement of florescent compounds (e.g., proteins) within thecell. These detection systems may be used to observe many differentchanges in internal cell activities in response to contact by compoundsfrom a library with the cell's membrane or receptors. These activitiesand similar activities are referred to herein collectively as “cellactivity,” and the apparatus employed to perform the assays are referredto herein as “cell activity assay apparatus.”

One kind of single-site cell activity assay apparatus referred tovariously in the literature as “chemotaxis chambers,” “Boyden chambers,”“Boyden chemotaxis chambers,” and “blind-well chambers,” may have twocompartments separated by a membrane, with one or both of thecompartments open to air. Multi-site apparatus, often referred to as“multi-well chemotaxis chambers” or “multi-well Boyden chambers,” mayhave the same basic site structure but have multiple sites. See, e.g.,U.S. Pat. Nos. 5,210,021 and 5,302,515, which are herein incorporated byreference in their entirety.

Assays employing this kind of apparatus may pipette cells suspended inmedia into the upper compartments, and pipette chemotactic factors andcontrols into the bottom compartments. The chemotactic factors may beused in various dilutions to get a dose-response curve. The controls aregenerally of three kinds: (a) negative, when the same media that is usedto suspend the cells is also used below the membrane; (b) chemokinetic,when a chemotactic factor is placed at the same concentration in themedia with the cells and in the well on the opposite side of themembrane; and (c) positive, when a known chemoattractant is placed inthe bottom wells. Chemokinetic controls may allow the user todistinguish heightened random activity of the cells, due to contact withthe chemotactic factor, from directional response in a concentrationgradient of that chemotactic factor.

Cell activity assay apparatus may also be used to measure the responseof cells of different origins, e.g., immune cells obtained from patientssuffering from diseases, to a compound with known chemotactic activity.In this case, the cells in question are interrogated by both a negativecontrol and a known chemotactic factor to see if the differentialresponse is depressed or normal. Traditionally, chemotactic activity hasbeen measured by establishing a stable concentration gradient in thecell activity assay apparatus, incubating it for a predetermined time,and then counting the cells that have migrated through the membrane (orinto the membrane). A comparison is then made between the activity ofthe cells in a concentration gradient of the chemotactic factor beingtested, and the activity of the cells in the absence of theconcentration gradient.

In one type of cell activity assay apparatus and method, the chemotacticresponse may be measured by physically counting the number of cells onthe membrane surface closest to the chamber containing the chemicalagent. An example of this type of cell activity assay apparatus isdescribed in U.S. Pat. No. 5,210,021, which is herein incorporated byreference. One method of obtaining quantitative data is to remove themembrane from the cell activity assay apparatus, remove the cells fromthe membrane surface closest to the chamber containing the original cellsuspension, fix and stain the remaining cells, and then observe andcount the stained cells under a microscope. Because of the time andexpense associated with examining the entire membrane, onlyrepresentative areas of the membrane may be counted, which may renderresults less accurate than would otherwise be the case if the entiremembrane were examined and counted.

Cell activity assays using a disposable ninety-six well microplateformat, for example, the ChemoTx™ System (available from Neuro Probe,Inc., Gaithersburg, Md.), may be amenable to different methods ofquantification of results. The manual staining and counting methoddescribed above can be used, but is not recommended due to the timeinvolved. A preferred method is to centrifuge the microplate with thefilter attached such that the cells that have migrated through thefilter are deposited onto the bottom of the lower wells. The cells maythen be stained with MTT, MTS (available from Promega, Madison, Wis.),or a similar dye, and then read in a standard automated laboratorydensitometric reader (sometimes referred to as an Elisa plate reader).

Another method of obtaining quantitative data with this type ofapparatus is to dye the cells with a fluorescent material, e.g., CalceinAM (available from Molecular Probes, Eugene, Oreg.), centrifuge themigrated cells into the microplate, and count cells with an automaticfluorescence plate reader (e.g., Cytofluor available from PE Biosystems,Foster City, Calif.; Victor2 available from EG&G Wallac, Gaithersburg,Md.; or fmax available from Molecular Devices, Sunnyvale, Calif.). Theautomatic plate reader excites the fluorescent dye in the migrated cellswith one wavelength of light and reads the light emitted at a secondwavelength. Alternatively, the cells that have not migrated may beremoved from the top of each site, and the plate with the framedmembrane attached may be read in the automatic fluorescent plate readerwithout spinning the cells into the plate, thereby counting the cellsthat have fallen off the filter into the lower well as well as those onthe bottom of the membrane and in the pores of the membrane.

As described above, past efforts at measuring chemotactic activity havefocused on measuring or counting cells that have passed through a long,tortuous path, such as through a filter or thick membrane. Because oftheir dependence on cell migration, these techniques may presentsignificant drawbacks. First, the cells must migrate a considerabledistance through the media to the chemotactic factor, which may addsubstantial time to the assay. Second, to obtain desirable (low)coefficients of variation, a relatively large number of cells is neededto calculate percentages of migration. Consequently, these assays demandlarge volumes of compound from a compound library, which are not alwaysreadily available. Third, in migration assays that count the number orpercentage of cells that have passed through a filter, the resultsprovide quantitative data, but not kinetic data. In addition, theresults provide no information about the cells that have not passedthrough the filter.

In contrast to migration assays, the present embodiments may morequickly determine cell activity by detecting changes in the cells thatoccur well before migration. Those changes may include, for example,cell orientation, internal morphological changes, temperaturevariations, molecular movement within the cell, and electromagneticchanges.

To provide that early detection, embodiments may provide a side sourcefrom which a test compound solution diffuses into a cell suspensionmedia and contacts cells. A side source may be an opening positioned tothe side of a test site, which extends substantially across the width ofthe test site, and from which a test compound solution diffuses in asubstantially linear fashion into a cell suspension media (e.g., movingsubstantially in a line or as a substantially linear “front”), away fromthe side source, and across the test site, and contacts cells within thetest site. Since the side source extends substantially across the widthof the test site, diffusion from the side source on one side of the testsite may be substantially linear across the test site. In embodiments,the side source extends across substantially the entire width of thetest site. For example, a test site may have an elongated shape (e.g.,oval shape) when viewed from a plan view, and the side source may extendacross nearly the entire width of the test site, as described inembodiments herein.

The present embodiments may also provide an apparatus having a sidesource, a point source, or both, from which a test compound solution maydiffuse into a cell suspension media and contact cells therein. The term“point” as used herein in the expression “point source” is not intendedto be a geometric term, but a relative term referring to a center areafrom which a compound diffuses concentrically. Since the area of a pointsource in this usage is relatively small with respect to the area of theentire site (e.g., less than 10% of area of the entire site), diffusionfrom the center area into the rest of the site is experimentallyequivalent to diffusion from a point. As an example, the concentricdiffusion may be readily observed from a top or bottom view of a site,with progressive rings extending outwardly from the point source incircles or in portions of circles (e.g., semicircles or quarter circles)depending on the configuration of the site and the location of the pointsource. Examples of point source diffusion are described in U.S. Pat.Nos. 7,547,525, 8,129,175, and 8,486,655, which are herein incorporatedby reference in their entirety.

In some of the present embodiments, a side source may be one type of apoint source, based on the size and location of the point sourcerelative to the configuration of a test site. As described above,diffusion from a point source may be in portions of circles depending onthe configuration of the site and the location of the point source.Thus, for example, a side source may be provided by a point sourcelocated at a side of a test site that is long and narrow relative to thesize of a point source. In that configuration, the partial-circlediffusion may be substantially linear along the long and narrow testsite.

The present embodiments may control the rate of this diffusion so thatcell activity can be progressively monitored. In one aspect of thepresent embodiments, the rate of diffusion may be gradual so that thecell activity caused by the test compound solution occurs in stages asthe test compound solution diffuses farther from the side source, sothat periodic readings of the site may detect such progressive changes.Embodiments may control the rate of diffusion using, for example, a gellayer occluding an opening through a top plate, a gel containing testcompound solution, a sintered material containing test compoundsolution, a frozen test compound solution, a dried or freeze-dried testcompound solution, and combinations thereof. One embodiment may providea method for determining whether a test compound solution induces cellactivity comprising placing the test compound solution in contact with acell suspension media containing cells, diffusing the test compoundsolution into the cell suspension media from a side source, a pointsource, or both, and detecting activity in the cells with respect totheir distance from the side source, point source, or both. Detectingactivity in the cells may involve detecting activity in a first group ofthe cells proximate to the side source, point source, or both, anddetecting activity in a second group of the cells farther from the sidesource, point source, or both, than the first group.

Another embodiment may provide a cell activity assay apparatus thatincludes a top and bottom plate, and an opening through the top plate,through which cell suspension media may be disposed on the top side ofthe bottom plate proximate to a side aperture leading to a cavitybetween the two plates, into which the suspension may spread. The testcompound solution may then be disposed on the top side of the bottomplate next to a side aperture leading to the cavity between the twoplates. In this configuration, the test compound solution contacts thecell suspension media within or proximate to the side aperture, anddiffuses into the cell suspension media, creating a concentrationgradient emanating from the side aperture into the cell suspensionmedia.

In one embodiment, there may be multiple openings to the cavity, one ofwhich is a small hole, and the concentration gradient may extendconcentrically from this opening. The cells nearest the opening may becontacted first by the test compound solution. Subsequently, as the testcompound solution diffuses farther from the opening, the other cells maybe contacted radially outward in stages. If the cells are responsive tothe test compound solution, the progressive diffusion may provideprogressive cell activity that, when monitored with suitable detectionmeans, yields both quantitative and kinetic (e.g., changes in the cellsas a function of time) data on cell activity.

An embodiment may provide a method for performing a cell activity assayusing the apparatus described above. According to this method, a cellsuspension may be deposited onto the top side of a bottom plateproximate to a side aperture leading to a cavity formed by the topsurface of the bottom plate and a bottom surface of a top plate, intowhich cavity the suspension may spread. The apparatus may then beincubated, which may allow cells to settle out of the cell suspensionand adhere to the top side of the bottom plate, leaving cells adhered tothe top side of the bottom plate and cell suspension media covering thecells. Optionally, the apparatus may then be “read” to provide abaseline reading of the cells' morphology and activity. “Read” in thiscontext may involve acquiring a high resolution picture (digital orotherwise) with, e.g., a high content screening detection instrument.The test compound solution may then be deposited on the top surface ofthe bottom plate proximate to a side aperture leading to a cavity—formedby the top surface of the bottom plate and a bottom surface of a topplate—that is occupied by the cell suspension. The test compoundsolution may be deposited, for example, by pipetting onto the top sideof the bottom plate, by a pin applicator, by projecting the testcompound solution down onto the top surface of the bottom plate, or byother appropriate means. With the test compound solution deposited, thetest compound solution may be in contact with the cell suspension mediaat the side aperture of the cavity. With this contact, the test compoundsolution may begin diffusing into the cell suspension media. The sitemay then be read periodically to observe the effect of the test compoundsolution on the cells as this diffusion progresses. These readingsdetect, for example, cell elongation and orientation, othermorphological changes, temperature changes, movement of molecules andstructures within a cell, and electromagnetic changes. Detectingmorphological changes involves, for example, examining the aspect ratio(length to width ratio) of the cells. Detecting cell orientationinvolves, for example, examining the orientation of the aspect ratio inrelation to the side aperture from which the test compound solution isdiffusing.

Alternatively, the apparatus described above may also be used first inassay development to determine the optimal time to perform a singledetection step, and then in the screening stage (which is typically theexpensive stage in time and materials) with a single detection orreading step. The assay development stage may use multiple detectionsteps—it may create a “movie” of the cells responding through time—whichdemonstrates and records the kinetics of the process. From this data, anoptimal time may be determined for performing a single detection orreading step in the actual screen. The single detection step may be at atime when the test compound has diffused across the site or part wayacross the site.

Thus, there will be many geometrically distinct subpopulations of cellsin the site at that time: at one end, proximate to the side source ofthe test compound, there is a subpopulation that has been exposed to thetest compound the longest duration and at the other end, farthest fromthe side source of the diffusion, is a subpopulation that has beenexposed the least duration. Typically, the optimal time to acquire thesingle detection step may be when the subpopulation at one end of thesite shows no change in cell activity, the subpopulation at the otherend of the test site (which has been exposed the longest) shows thelargest alteration in its activity, and the subpopulations in betweenshow a continuum of change in cell activity from a lot to a little tonone at the end of the site farthest from the side source.

Furthermore, the exposed subpopulation has been exposedprogressively—those closer to the side source exposed longer than thosefarther away. Thus, even though there is a single detection step, theactivity, non-activity, and extent of activity of the cells will expressthe kinetics of the situation. In this manner, the present embodimentscan detect activity in the cells with respect to their distance from theside source.

Abbreviations & Definitions

1. “High throughput screening” is herein abbreviated to “HTS.”

2. “Cell-based high throughput screening” is herein abbreviated to“CBHTS.”

3. “High Content Screening” is herein abbreviated to “HCS.”

4. “Nanometer” is herein abbreviated to “nm.”

5. “Microliters” is herein abbreviated to “ul.”

6. “Micrograms” is herein abbreviated to “ug.”

7. “Test compound solution” as used herein may refer to a solutioncomposed of a compound of interest to a researcher, for example, acompound from a compound library, dissolved in water, cell culturemedia, dimethyl sulfoxide (DMSO), other appropriate media, or acombination thereof.

8. “Cell suspension media” as used herein may refer to a media or fluidcapable of suspending cells.

9. “Cell suspension” as used herein may refer to a solution containingcells suspended in a cell suspension media.

10. “Point source” as used herein may refer to a center area from whicha chemical compound diffuses concentrically.

11. “Side source” as used herein may refer to a side aperture or openingof a cavity in an assay platform from which an aqueous solution canenter and a chemical compound solution can diffuse. It may be a type ofpoint source.

12. “Mechanical hydrophobic barrier” as used herein may refer to aphysical feature of an assay apparatus (e.g., a groove in the bottom ofa plate with sharp right angles) that, when held against a flat surfaceof a second plate, creates a barrier to aqueous solutions and preventstheir passing without significant hydrostatic pressure being applied.

13. “Chemical hydrophobic barrier” as used herein may refer to achemical feature of an assay apparatus, e.g., a chemical compoundapplied to a plate, or a film incorporating a chemical compound that isheld against a (flat top) surface of a plate, that creates a barrier toaqueous solutions and prevents their passing without significanthydrostatic pressure being applied.

14. “Read” as used herein may refer to the capture of data from thesites of a cell activity assay apparatus, which may include theacquisition of images, digital or otherwise, from each site of the cellactivity assay apparatus.

Other systems, methods, features, and advantages of the embodiments willbe, or will become, apparent to one of ordinary skill in the art uponexamination of the following figures and detailed description. It isintended that all such additional systems, methods, features, andadvantages be included within this description and this summary, bewithin the scope of the embodiments, and be protected by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments can be better understood with reference to the followingdrawings and description. The components in the figures are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the embodiments. In addition, in the figures, likereference numerals designate corresponding apparatus parts throughoutthe different views.

FIG. 1.1 is a schematic diagram of a cross-section of one site of anembodiment of a multi-site assay apparatus, taken along line 1.1-1.1 ofFIG. 1.4, having a top plate with a single through-well, a transparentbottom plate, a mechanical or geometric hydrophobic barrier enclosing ablind cavity between the top and bottom plate, and clamping componentsto clamp the top and bottom plates together.

FIG. 1.2 is a schematic diagram of a cross-section of one site ofanother embodiment of a multi-site assay apparatus, having a top platewith a single through-well, a transparent bottom plate, a chemicalhydrophobic barrier enclosing a blind cavity between the top and bottomplates, and clamping components to clamp the top and bottom platestogether.

FIG. 1.3 is a schematic diagram of a cross-section of one site ofanother embodiment of a multi-site assay apparatus, having a top platewith a single through-well, a transparent bottom plate, a mechanical orgeometric hydrophobic barrier and a chemical hydrophobic barrier whichtogether enclose a blind cavity between the top and bottom plates, andclamping components to clamp the top and bottom plates together.

FIG. 1.4 is a schematic diagram of a top view of one site of themulti-site assay apparatus of FIG. 1.1, having a top plate with a singlethrough-well, a transparent bottom plate, and a mechanical or geometrichydrophobic barrier enclosing a blind recessed cavity between the topand bottom plates, with a schematic representation of a concentrationgradient of a chemoattractant test compound and subpopulations of cellsresponding within the test site, according to an embodiment.

FIG. 1.5 is a schematic diagram of an enlarged cross-sectional view of adetail of the site shown in FIG. 1.4 of the multi-site assay apparatusof FIG. 1.1, which has a top plate with a single through-well, atransparent bottom plate, and a mechanical or geometric hydrophobicbarrier enclosing a blind cavity between the top and bottom plates, witha schematic representation of a concentration gradient of achemoattractant test compound and of cells responding within the testsite, according to an embodiment.

FIG. 1.6 is a schematic diagram of a further enlargement of FIG. 1.5,showing a detail of the mechanical or geometric hydrophobic barrier atthe interface between the top plate and the bottom plate. Here thebarrier encloses one end of the blind cavity between the two plates,along with a schematic representation of a cell, according to anembodiment.

FIG. 1.7 is an isometric semi-transparent view of the site of themulti-site assay apparatus of FIGS. 1.1 and 1.4.

FIG. 2.1 is a schematic diagram of a cross-section of one site ofanother embodiment of a multi-site assay apparatus, taken along line2.1-2.1 of FIG. 2.3, having a top plate with two through-wells, atransparent bottom plate, a mechanical or geometric hydrophobic barrier,and a recessed cavity between the top and bottom plates.

FIG. 2.2 is a schematic diagram of a cross-section of one site ofanother embodiment of a multi-site assay apparatus, having a top platewith two through-wells, a transparent bottom plate, a chemicalhydrophobic barrier, and a non-recessed cavity between the top andbottom plates.

FIG. 2.3 is a schematic diagram of a top view of the assay apparatus ofFIG. 2.1, showing a schematic concentration gradient in the gel-filledcavity and cells in a well responding and moving into the cavity,according to an embodiment.

FIG. 2.4 is a schematic diagram of a top view of one site of anotherembodiment of the multi-site assay apparatus of FIG. 2.1, having a topplate with two through-wells and a mechanical or geometric hydrophobicbarrier, and a test compound in cell culture media in one well, showinga schematic concentration gradient in the gel-filled central cavity,with cells responding within the test site, according to an embodiment.

FIG. 3.1 is a schematic diagram of a cross-section of one site of amulti-site assay apparatus, taken along line 3.1-3.1 of FIG. 3.2, havinga top plate with a through-well at each end of the site, a small taperedcenter through-hole, a transparent bottom plate, a cavity between thetop and bottom plates, and two hydrophobic barriers, one mechanical andthe other chemical, according to an embodiment.

FIG. 3.2 is a schematic diagram of a top view of one site of themulti-site assay apparatus of FIG. 3.1, having a mechanical hydrophobicbarrier, and illustrating a schematic concentration gradient in thegel-filled central cavity, with cells responding within the test site,according to an embodiment.

FIG. 4.1 is a schematic diagram of a top view of an embodiment of amulti-site assay apparatus, having a through-well at the left and rightends of the test site, a middle well with a tapered through-hole, arecessed cavity, and a mechanical hydrophobic barrier, with a schematicconcentration gradient and with cells responding within the test site,according to an embodiment.

FIG. 4.2 is a schematic diagram of a top view of the multi-site assayapparatus of FIG. 4.1, with a schematic concentration gradient comingfrom a piece of tumor or other tissue in the left well, and with cellsresponding within the test site, according to an embodiment.

FIG. 4.3 is a schematic diagram of a cross-section of the assayapparatus of FIG. 4.4, taken along line 4.3-4.3 of FIG. 4.4, showing afirst cell suspension in the left well with a test compound thatinhibits migration, a second cell suspension in the right well without atest compound, and a known chemoattractant in the middle well diffusingconcentrically out across the cavity between top and bottom plates, withcells from the right well migrating into cavity, and cells from the leftwell not migrating, according to an embodiment.

FIG. 4.4 is a schematic diagram of a top view of the assay apparatus andcells of FIG. 4.3, showing a schematic representation of a first cellsuspension in the left well with a test compound that inhibitsmigration, a second cell suspension in the right well without a testcompound, and a known chemoattractant in the middle well diffusingconcentrically across the cavity between the top and bottom plates, withcells from the right well migrating into cavity, and cells from the leftwell not migrating, according to an embodiment.

FIG. 4.5 is a schematic diagram of a cross-section of the assayapparatus of FIG. 4.6, taken along line 4.5-4.5 of FIG. 4.6, which is amodified version of the assay apparatus of FIG. 4.1, having athrough-well at each of the left and right ends of a test site, a middlewell with a tapered through-hole, a relatively deep, hence enlarged,recessed cavity, and a mechanical hydrophobic barrier, with a piece oftumor or other tissue in the left well, with an enlarged (deeper) cavitybetween the top and bottom plates, and with cells shown before aconcentration gradient of compounds from the tumor or other tissue hasformed, according to an embodiment.

FIG. 4.6 is a schematic diagram of a top view of the assay apparatus ofFIG. 4.5, showing a schematic representation of a piece of tumor orother tissue in the left well, which is open to an enlarged (deeper)cavity between the top and bottom plates, and showing cells in thecavity before a concentration gradient of compounds from the tumor orother tissue has formed, according to an embodiment.

FIG. 4.7 is a schematic diagram of a cross-section of the assayapparatus of FIG. 4.1, having two through-holes, a tapered middle wellwith a small through-hole, a recessed cavity, and a mechanicalhydrophobic barrier, with a piece of tumor or other tissue in the middlewell, cell monolayers in the left and right wells, and cell suspensionswith cells above the cell monolayers in the left and right wells,according to an embodiment.

FIG. 4.8 is a partially transparent isometric view of the multi-siteassay apparatus of FIG. 4.1, according to an embodiment.

FIG. 4.9 is a cross-sectional isometric view of the multi-site apparatusof FIG. 4.1, taken along line 4.9-4.9 of FIG. 4.8, according to anembodiment.

FIG. 5 is a top view schematic diagram of an embodiment of a singlefive-well site of a multi-site assay apparatus, having a top plate withfive through-wells, a transparent bottom plate, and a mechanical orgeometric hydrophobic barrier enclosing a cavity between the top andbottom plates, according to an embodiment.

FIG. 6.1 is a schematic diagram of a top view of another embodiment of amulti-site assay apparatus, having multiple wells enclosing a test site.

FIG. 6.2 is a partial cross-sectional isometric view of the multi-siteapparatus of FIG. 6.1, taken along line 6.2-6.2 of FIG. 6.1, accordingto an embodiment.

FIG. 6.3 is a partial cross-sectional isometric view of the multi-siteapparatus of FIG. 6.1, taken along line 6.3-6.3 of FIG. 6.1, accordingto an embodiment.

DETAILED DESCRIPTION

Embodiments provide methods and systems for placing a test compoundsolution in contact with a cell suspension media containing cells,diffusing the test compound solution into the cell suspension from oneor more sides, and detecting activity in the cells with respect to theirdistance from the side from which the test compound is diffusing.

In embodiments of side source or point source diffusion cell activityassays, a test compound may be introduced so as to initiate a stablediffusion gradient of that compound through a cell population or cellpopulations—to develop a diffusion gradient that is stable over a periodof time. Stability in this context implies that there is no significantflow of the fluid media surrounding the cells. That lack of flow mayallow detection of cell activity caused or initiated by interactionbetween the test compound and cells in the cell suspension according totheir distance from the side source or point source from which diffusionof the test compound begins. A single image of the field of affectedcells may then be captured after a specific period of time (e.g.,determined in assay development) to document cell activity and thedynamics of cell activity (virtual kinetics). To accomplish this, theapparatus may prevent flow in the media that surrounds the cells for theperiod of time required for the diffusion gradient to be establishedacross that field.

In one side source diffusion embodiment, the fluid of the cellsuspension is constrained by both mechanical and hydrophobic barriers.The cell suspension may completely fill the site or sites of theapparatus before the introduction of the test compound solution, andconsequently there may be no space into which the cell suspension canflow. In other embodiments disclosed herein, the cell suspension may notcompletely fill the site or sites of the apparatus, and fluid flow maybe prevented by the use of a gel or gels, either in the cell suspensionor next to the cell suspension. The gel may prevent the flow of fluid,but permit the establishment of a concentration gradient.

The embodiments illustrated in FIGS. 1.1-1.7, namely those with a singlewell or through-opening into the cavity 108, may not involve theemployment of a gel in the cell suspension media, since the introductionof test compounds in aqueous solution does not cause flow of the cellsuspension media in the cavity. There is nowhere for the cell suspensionmedia to flow.

FIG. 1.1 illustrates a cross-sectional view of one site of an embodimentof a multi-site cell activity assay apparatus 100. As shown, assayapparatus 100 may include a top plate 101 (which may also be referred toas a cover) with a through-well opening 102 to the top surface 109 of atransparent bottom plate 107 (which may also be referred to as asubstrate). The bottom surface 110 of top plate 101 may have a machinedor otherwise formed recessed area 113. The top plate 101 may thereforehave two surfaces facing the bottom plate 107: the recessed surface 113and the remaining surface area of the bottom surface 110 of top plate101. When the top and bottom plates are pressed together, bottom surface110 may be in contact with top surface 109, but there may be a space—acavity 108—between top surface 109 and recessed surface 113. Around theperimeter of cavity 108 there may be a groove 106 with a sharp corner(e.g., 90 degree corner) that creates a mechanical hydrophobic barrier105 to fluids in cavity 108. The volume of cavity 108 may be determinedby the depth of the recessed surface 113 in the bottom surface of topplate 101.

In an embodiment, the mechanical hydrophobic barrier 105 is not apneumatic seal; thus, an aqueous solution can fill the cavity 108between surfaces 113 and 109 by capillary action, the gas in cavity 108being displaced by the aqueous solution and exiting through thehydrophobic seal provided by the mechanical hydrophobic barrier. Whenthe cavity 108 is full of a fluid or gel, a test compound solutionintroduced into the through-well 102 may contact the fluid or gel andbegin diffusing out into it from the gap between the bottom edge ofthrough-well 102 and surface 109. That gap is the side source 112 of theconcentration gradient that ensues in cavity 108. Referring to FIGS.1.1, 1.4, and 1.7, the gap may extend substantially across the width ofthe test site (e.g., defined by the hydrophobic barrier), for example,within a range from over 50% of the width of the test site to 100% ofthe width of the test site. For example, the bottom edge of through-well102 may span nearly the entire width of the test site, betweenhydrophobic barrier 105. In one embodiment, referring to FIG. 1.4, atest site may have a width of approximately 5 mm between the hydrophobicbarrier 105, and a through-well 102 may have a diameter of approximately3 mm. In another embodiment, a test site may have a width ofapproximately 6 mm between the hydrophobic barrier 105, and athrough-well 102 may have a diameter of approximately 5 mm. In yetanother embodiment, the width of the test site between the hydrophobicbarrier 105 and the diameter of a through-well 102 may be substantiallyequal. The hydrophobic seal provided by the mechanical hydrophobicbarrier 105 may ensure that cavity 108 is a “blind cavity” in the sensethat through-well 102 is the only opening through which fluids can enteror exit cavity 108. The top plate 101, the hydrophobic barrier 105, andthe bottom plate 107 may be held together to make and sustain contactwith one another, for example, as depicted schematically in FIG. 1.1, byclamp 130 and 131. Other fasteners may be used to secure the componentsincluding, for example, threaded fasteners and adhesives.

FIG. 1.2 illustrates a cross-sectional view of one site of anotherembodiment of a side-source multi-site cell activity assay apparatus103. Apparatus 103 differs from apparatus 100 of FIG. 1.1 in that thebottom surface 110 of top plate 101 has no recessed area, and thehydrophobic barrier is a chemical hydrophobic barrier 111 rather than amechanical one. The chemical hydrophobic barrier 111 may be printed orotherwise applied to the top surface 109 of the bottom plate 107. Whenthe top plate 101 is held in contact with bottom plate 107 (e.g., usingclamp 130 and 131), a cavity 108 is formed by surfaces 109 and 110,along with the chemical hydrophobic barrier 111. The distance betweensurfaces 109 and 110, and therefore the volume of cavity 108, may bedetermined by the thickness of the hydrophobic barrier 111.

As in FIG. 1.1, in an embodiment, the hydrophobic barrier 111 is not apneumatic seal, and an aqueous solution may fill cavity 108 by capillaryaction, displacing gases, which can exit through the hydrophobic barrier111. When cavity 108 is full of a fluid or gel, a compound solutionintroduced into the through-well 102 may contact the fluid or gel andbegin diffusing out into it from the gap between the bottom edge ofthrough-well 102 and the surface 109. That gap is the side source 112 ofthe concentration gradient that ensues in cavity 108. The hydrophobicseal provided by the hydrophobic barrier 111 may ensure that cavity 108is a blind cavity in the sense that through-well 102 is the only openingthrough which fluids can enter or exit cavity 108.

FIG. 1.3 illustrates a cross-sectional view of one site of anotherembodiment of a side-source multi-site cell activity assay apparatus193. Apparatus 193 differs from apparatus 100 of FIG. 1.1 and apparatus103 of FIG. 1.2 in that apparatus 193 has both a mechanical hydrophobicbarrier 105 and a chemical hydrophobic barrier 111. The bottom surface110 of top plate 101 may have no recessed area; in that case, the volumeof blind cavity 108 may be determined by the thickness of thehydrophobic barrier 111.

FIG. 1.4 is a schematic diagram of a top view of one site of theside-source multi-site cell activity assay apparatus 100 of FIG. 1.1,according to an embodiment. FIG. 1.4 shows the top plate 101 with thesingle through-well 102, part of the top surface 109 of the bottom plate107, and a mechanical hydrophobic barrier 105 enclosing a blind cavity108 between the top plate 101 and the bottom plate 107. An oval grooveor channel 106 formed in top plate 101 surrounds the site and, with itssharp angle (e.g., right angle) intersection with surface 110, forms amechanical hydrophobic barrier 105 around blind cavity 108. In cavity108 within the hydrophobic barrier 105, FIG. 1.4 shows a schematicrepresentation (dashed lines 114 through 119) of a concentrationgradient diffused across cavity 108 starting from side source 112 asrepresented by arrows 199, with the most concentrated part of thegradient being near side source 112 and represented by line 114, andwith the least concentrated part of the gradient being farthest fromside source 112 and represented by line 119.

FIG. 1.4 also shows cells (121 through 126) in a cell suspension 120filling cavity 108, with the most responsive cells 121 located closestto the side source 112 and the least responsive cells 126 locatedfarthest from the side source 112. Typically, the concentration gradientwould be created by introducing a test compound, positive controlsolution, or other fluid of interest into the through-well 102 at theside source 112 and allowing the fluid to diffuse through the cellsolution 120 in cavity 108.

FIG. 1.4 schematically represents a reading of the site after theconcentration gradient has formed and the cells in the blind cavity 108have responded by elongating and migrating toward the side source 112 ofthe concentration gradient. The shapes of the cells in these schematicrepresentations are typical of immune cells responding to achemoattractant. For example, the cells closest to the side source 112of the chemoattractant react first and therefore have a higher aspectratio (i.e., are more elongated.) However, here the cell shapessymbolically represent any morphological or other detectable (e.g.,visually detectable) changes in the cell population, including internalmorphological changes. By selecting representative cells at intervalsacross the cavity 108 and analyzing the morphology or other changes inthe cells, the kinetics of the cell activity in response to the testcompound may be inferred.

FIG. 1.5 illustrates an enlarged cross-sectional view of a detail of theside-source multi-site cell activity assay apparatus 100 illustrated inFIG. 1.1 and FIG. 1.4, according to an embodiment. As in FIG. 1.1, thetop plate 101 may have a recessed area 113 and an oval groove 106. Apartfrom the groove 106, top plate 101 may have two surfaces facing bottomplate 107: the recessed area 113, which faces but does not contact thetop surface 109 of the bottom plate 107, and the portion of the bottomsurface 110 of top plate 101 that is not recessed or grooved and thus isin contact with surface 109 when the top and bottom plates are heldtogether. As in FIG. 1.1, top plate 101 and bottom plate 107 may formthe volume of cavity 108, the cavity 108 may be a blind cavity, and thehydrophobic barrier 105 may permit passage of gas.

Like FIG. 1.4, FIG. 1.5 shows a cell suspension 120 containing cells121, 122, 123, 124, 125, 126. In an embodiment, the cell suspension 120may be deposited on the top surface 109 of bottom plate 107 proximate tothe side source 112 and into the cavity 108, and may fill out the cavity108 to the hydrophobic barrier 105 and form a meniscus 127 at theopening of side source 112. A test compound, positive control, or otherfluid of interest 140, placed in through-well 102, may contact the cellsuspension 120 at the meniscus 127 at side source 112, and may diffuseout through the cell suspension 120, creating a concentration gradient,which is represented by lines 114, 115, 116, 117, 118, 119. The compound140 may be most concentrated near side source 112, with lines 114, 115,116, 117, 118, 119 representing progressively weaker concentrations ofthe compound 140 moving away from the side source 112. As represented inFIG. 1.5 by the differently shaped cells 121, 122, 123, 124, 125, 126,the cells 121, 122, 123, 124, 125, 126 may progressively respond tocontact with the compound 140, with the strongest response at cell 121where the concentration is highest, and the weakest response at cell 126where the concentration is lowest.

FIG. 1.6 illustrates a further magnified schematic cross-section of adetail of the side-source multi-site cell activity assay apparatus 100illustrated in FIG. 1.1 and FIG. 1.5, according to an embodiment. Thisdetail illustrates an end portion of cavity 108 that is farthest fromthe side source 112 of the concentration gradient. As shown, the cellsuspension 120 may move beyond the end of cavity 108 until the cellsuspension 120 reaches the hydrophobic barrier 105, where the cellsuspension 120 may form a meniscus 128 and stop moving. The fluid of thecell suspension 120 will breach the hydrophobic barrier 105 only ifsubstantial hydrostatic pressure is applied. Since the cell suspension120 may completely fill cavity 108 and there are no openings throughwhich the cell suspension 120 may move, no further flow of the cellsuspension 120 can occur.

FIG. 1.7 is a semitransparent isometric view of one site of theside-source multi-site assay apparatus 100 that is illustrated in FIGS.1.1 and 1.4, according to an embodiment.

In an exemplary implementation of the embodiments illustrated in FIGS.1.1-1.5, a single site may be part of a 48-site apparatus, in which eachsite may be approximately 9 mm×18 mm and the area within each site,defined by its hydrophobic barrier, may be approximately 7 mm×16 mm,with a through-well having a diameter of about 6 mm. In anotherembodiment, each site may be 6 mm×18 mm, and the area within each sitemay be oval shaped and approximately 5 mm×16 mm, with a through-wellhaving a diameter of about 3 mm. Bottom plate 107 can be glass or aplastic material or other transparent material such as sapphire. Topplate 101 can be a plastic material, e.g., acrylic, or other suitablematerial.

Although figures herein illustrate a single assay or test site, one ofordinary skill in the art can appreciate that the illustrated structurescould be replicated in multiple-site apparatus, for example, apparatushaving 24, 32, 48, 96, 192, 364, 768, or 1536 sites.

An embodiment provides a method for performing a cell activity assayusing the exemplary apparatus 100 shown in FIGS. 1.1 and 1.4-1.6.According to this method, a cell suspension 120 (containing cellssuspended in a cell suspension media) may be deposited onto the topsurface 109 of the bottom plate 107 and next to the side source 112between the top surface 109 of bottom plate 107 and the recessed surface113 of the top plate 101. The cell suspension 120 may then fill thecavity 108 between surfaces 113 and 109 and bounded by the hydrophobicbarrier 105. The apparatus may then be incubated, which allows the cells121 through 126 to adhere to the top side 109 of the bottom plate 107.The sites may then be read and recorded to provide a baseline reading ofcells, or this step can be done immediately after the test solution isintroduced.

The test compound solution 140 may then be deposited on the top surface109 of bottom plate 107 next to the side source 112 between top surface109 of bottom plate 107 and recessed surface 113 of top plate 101. Thetest compound solution can be deposited, for example, by pipetting, by apin applicator, or by other appropriate means.

With test compound solution 140 deposited on the top surface 109 of thebottom plate 107, and next to side source 112, the test compoundsolution 140 may be in contact with cell suspension 120 at the sidesource 112. With this contact, test compound solution 140 may begindiffusing into the media of cell suspension 120. The site may then beread periodically to observe the effect of the test compound solution140 on the cells 121-126 as this diffusion progresses. These readingsmay detect, for example, cell elongation and orientation, othermorphological changes, temperature changes, movement of molecules andstructures within a cell, and electromagnetic changes. Detectingmorphological changes involves, for example, examining the aspect ratio(length to width ratio) of the cells. Detecting cell orientationinvolves, for example, examining the orientation of the aspect ratio inrelation to the side source 112 from which the test compound solution isdiffusing.

In the exemplary apparatus 100 shown schematically and magnified inFIGS. 1.5 and 1.6, cell suspension media 120 may fill the cavity 108,with a meniscus 127 formed around the bottom rim of through-well 102,i.e., at the side source 112. This configuration may provide an initialboundary (at meniscus 127) between the cell suspension 120 in the cavity108 and fluid in the through-well 102. In this example, the cellsuspension 120 may occupy the opening of side source 112 because thecell suspension 120 is deposited first at this opening with enoughvolume to just fill the cavity 108, wick by capillary action out tohydrophobic barrier 105, and form the meniscus 127 around the bottom ofthe through-well 102.

As the diffusion of the test compound solution 140 occurs, the testcompound reaches the cells 121 nearest the opening of side source 112first and then eventually, over time, reaches the cells 122-125, andthen cells 126 farthest away from the opening of side source 112. Duringassay development, apparatus 100 may be periodically read to determinewhether and when the cells 121-126 are responsive to the diffusiongradient of test compound solution 140. If the cells are responsive, theeffects are perceptible in stages, as the test compound solution 140diffuses generally linearly from side source 112. In this manner, theprogressive responses by cells 121-126 can yield both quantitative andkinetic data. From this data, the optimal time to acquire a single dataset or image of the cell activity can be determined, and can be usedsubsequently to acquire a single data set or image of the test sitesthat captures both quantitative and kinetic information about theeffects of the test compound on the cells in the cell suspension.

In the exemplary apparatus 100 shown in FIGS. 1.5 and 1.6, cellsuspension media 120 may fill the cavity 108, with a larger meniscus 127formed at the open aperture of side source 112 of cavity 108 and asmaller meniscus 128 formed at the mechanical hydrophobic barrier 105.This configuration may provide an initial boundary (at meniscus 127)between test compound solution 104 and cell suspension 120. In thisexample, the cell suspension 120 may occupy cavity 108 by beingdeposited at the side source 112, filling the cavity 108 by capillaryaction, and forming the meniscus 127 at the opening of side source 112.

Apparatus 100 may be periodically read as the diffusion continues andthe test compound solution 140 reaches distances farther from the sidesource 112, as represented by the gradient lines 114-119 in FIG. 1.4.When the apparatus 100 is read at these increments of diffusion, cellswithin the respective gradient lines may show a response (assuming thecells are responsive to the test compound solution). Moreover, all ofthe cells within the outermost gradient line that the test compoundsolution 140 has reached may show varying degrees of response. Forexample, if the test compound solution 140 has reached the fourthgradient line 117, the cells 121 within the second gradient line 115 mayshow the most response and the cells 123 between the third gradient line116 and the fourth gradient line 117 may show the least response, withthe cells 122 between the second gradient line 115 and the thirdgradient line 116 showing a response somewhere in between.

Each of FIGS. 2.1-2.4, 3.1-3.2, and 4.1-4.9 illustrates one site of amulti-site cell activity assay apparatus (200, 300, and 400respectively), according to other embodiments. Apparatus 200, 300, and400 are similar in most respects to the apparatus 100 illustrated inFIGS. 1.1-1.7, except that apparatus 200, 300, and 400 have a secondthrough-well or opening in the top plate. As shown in FIGS. 2.1-2.4,apparatus 200 includes through-wells 202 and 203 in top plate 201. Asshown in FIGS. 3.1-3.2, apparatus 300 includes through-wells 302 and 303in top plate 301, as well as a center small through-hole 304 through topplate 301. As shown in FIGS. 4.1-4.9, apparatus 400 includesthrough-wells 402 and 403, as well as a center well with a smallthrough-hole 404 in the bottom that goes through top plate 401. Theseembodiments and the related variations are topologically different fromapparatus 100 in that the cavities 208, 308, and 408 formed between thetop surfaces 209, 309, and 409 of the bottom plates 207, 307, and 407and the bottom surfaces 210, 310, and 410 (or the recessed portions 213,313, and 413 of the bottom surfaces) of the top plates 201, 301, and 401are not “blind” cavities; i.e., there are multiple ways for fluids toenter and exit these cavities.

FIGS. 2.1, 2.3, and 2.4 represent one site of apparatus 200, amulti-site side-source cell activity assay apparatus according to anembodiment. Sites of apparatus 200 may each have two through-wellopenings 202 and 203 in the top plate 201. Through these wells, fluids(e.g., cell suspensions, media, test compounds, positive controlsolutions, and other fluids of interest) may be introduced. A cavity 208may be provided between the top surface 209 of the transparent bottomplate 207 and, in FIG. 2.1, the recessed portion 213 of the bottomsurface 210 of the top plate 201. Alternatively, as shown in thealternate cell activity assay apparatus 250 in FIG. 2.2, the bottomsurface 210 of top plate 201 may have no recessed area, so cavity 208 isformed between surfaces 209 and 210. Cavity 208 may be bounded at itsperimeter by a hydrophobic barrier, either mechanical 205 (e.g., astructural corner of a groove or channel 206), as in FIG. 2.1, orchemical 211, or both, as in FIG. 2.2. The volume of cavity 208 may bedetermined by the distance between surfaces 213 and 209 in FIG. 2.1, andbetween surfaces 210 and 209 in FIG. 2.2. A hydrophobic barrier around asite may not be a pneumatic seal, so that gasses may escape from thecavity through the hydrophobic barrier. Cavity 208 is not a “blind”cavity, since there are two openings—through-wells 202 and 203—throughwhich fluids may enter and exit.

In FIGS. 2.1 and 2.2, a suitable quantity of a fluid, e.g., a liquidcell suspension, may be placed in the bottom of a through-well, such asthrough-well 202, at the side source 212, which may be the gap betweensurface 209 and the edge of the through-well 202 closest to the rest ofthe site. As shown in FIG. 2.3, the gap may extend substantially acrossthe width of the test site, for example, extending across nearly theentire width between hydrophobic barrier 205. In embodiments, the gapmay extend substantially across the width of the test site (e.g.,defined by the hydrophobic barrier), for example, within a range fromover 50% of the width of the test site to 100% of the width of the testsite, as described above in reference to FIGS. 1.1, 1.4, and 1.7. Thefluid suspension may move out from the side source 212 by capillaryaction into the cavity 208, filling the cavity 208 by spreading betweensurfaces 209 and 213 (FIG. 2.1), or 209 and 210 (FIG. 2.2), as far asthe hydrophobic barrier 205 (FIG. 2.1) or 211 (FIG. 2.2) surrounding thesite. The fluid of the cell-suspension media may breach hydrophobicbarrier 205 or 211 only if substantial hydrostatic pressure is applied,but, because of surface tension, the fluid may not move into theportions of surface 209 that form the bottoms of the through-wells 202and 203. The gap at the bottom edge of the well closest to the rest ofthe site—between surfaces 213 and 209 in FIG. 2.1, or between surfaces210 and 209 in FIG. 2.2—may provide a side source 212 from which a fluidcan move by capillary action to fill the cavity 208, or to diffuseacross the cavity 208 through a liquid or gel, forming a concentrationgradient.

FIGS. 2.1-2.4 illustrate methods of employing apparatus 200. In anembodiment, a method may first introduce into the cell suspension mediaa gelling compound in liquid phase that is temperature sensitive, pHsensitive, or sensitive to another change in conditions. The liquid cellsuspension media with gelling compound may then be introduced intoeither through-well 202 or 203 at the side source 212, and may fill thecavity 208, as described above. Since cavity 208 is not a blind cavity,as long as the cell suspension is in a liquid state, the cell suspensionmay flow within the cavity 208. After the media or cell suspension hasfilled cavity 208, flow may be prevented by allowing or inducing themedia to become a gel, e.g., by implementing appropriate changes inconditions that induce the media to gel.

In FIG. 2.3, which illustrates a top view of one site of the apparatus200 of FIG. 2.1, the initial introduction of cell suspension media 220contains no cells. The cell suspension media 220 moves into cavity 208by capillary action and fills the cavity 208 out to the hydrophobicbarrier 205 except for the portions of the surface 209 that form thebottoms of through-wells 202 and 203. The cell suspension media 220 maythen be induced to gel. A test compound 240 may be introduced intothrough-well 202, may contact the gelled cell suspension media 220 atside source 212, and may diffuse across cavity 208 starting from sidesource 212 as represented by arrows 299, forming a concentrationgradient represented by dashed lines 215, 216, 217, and 218. Line 215represents the most concentrated level of test compound 240, while line218 represents the least concentrated level of test compound 240. Cellsuspension containing cells may be introduced into through-well 203.When the diffusing test compound 240 reaches through-well 203, the cellsbegin to respond. For example, as shown in FIG. 2.3, the cells 221, 222,and 223 may elongate, orient, and migrate toward more concentratedlevels of test compound 240. As shown, cells 221, 222, and 223 mayexhibit increasing responses to increasing concentrations of testcompound 240 as the cells 221, 222, and 223 migrate across cavity 208.Data may be recorded at any point or points during this process for usein assay development, or as a reading during an assay.

FIG. 2.4 is another top view of one site of multi-site apparatus 200.Here, the site is prepared by introducing a cell suspension 220 (e.g.,including media and cells) at a side source 212 (e.g., the opening, orgap, at the bottom edge of one of the through-wells 202 and 203, whichmay extend substantially across the width of the test site). The cellsuspension 220 fills cavity 208 out to hydrophobic barrier 205, notflowing into the portions of surface 209 that form the bottoms ofthrough-wells 202 and 203. The cell suspension 220 may be induced togel, preventing any further flow within the cell suspension 220. Cellsuspension media without cells or gelling-compound may be introducedinto wells 202 and 203, the apparatus may be incubated for an optimalperiod (determined during assay development), after which a testcompound 240 may be introduced into well 203 (after removing any media).The test compound 240 diffuses starting from side source 212 asrepresented by arrows 299, and through the gelled cell suspension 220and across cavity 208, forming a concentration gradient, which isrepresented in FIG. 2.4 by the series of dashed lines 219, 218, 217,216, and 215. The concentration is greatest at line 219, and weakest atline 215. As the test compound 240 diffuses, the cells in the cavity maybegin to respond to the test compound 240, as represented by thedifferent shapes and elongations of cells 221, 222, 223, 224, and 225.Cells 221 are contacted first by the test compound 240, are exposedlongest, and experience the most concentrated level of test compound240. Cells 224 respond to the weakest concentration of test compound240, and for the least amount of time. The test compound 240 has notreached cells at 225; thus, those cells are not responding. Here againdata can be recorded at any point or points in time during this processfor use in assay development, or as a reading during an assay.

FIGS. 3.1-3.2 and FIGS. 4.1-4.4 illustrate other embodiments ofside-source diffusion, including cell activity assay apparatus 300 and400, respectively. In those embodiments, two wells or openings 302, 303and 402, 403 may be provided through the top plate 301, 401, throughwhich cell suspension media and test solutions may be introduced, aswell as a third through-hole 304, 404 between the other two wells oropenings. The process to set up an assay with these embodiments issimilar to that described above in reference to FIG. 2.4 for the cellactivity assay apparatus 200, but differs after the cell suspensionfluid has gelled (by, for example, temperature change, PH change, orother changes in condition). Test solutions, media solutions, cellsuspensions, pieces of tissue, and sintered material containing testcompounds are some of the substances that can, without causing the cellsuspension to flow, be introduced using the three through-holes. Ifpoint source diffusion is desired, the test solution may be introducedthrough the center well through-hole 304, 404, such that the diffusiongradient forms concentrically from that through-hole. If the testsolution is introduced from either of the side wells 302, 303 or 402,403, the diffusion gradient develops from that side well, which isreferred to herein as a “side source” of compound diffusion. Note thatthe two- and three-well configurations 200, 300, 400 that use gel toprevent flow of the cell suspension not only enable cell activity assaysof a simple one-compound one-cell type, but may also be used fornumerous more complex assays employing multiple cell types and multiplecompounds.

As with the cell activity assay apparatus 100 and 200, the cell activityassay apparatus 300 and 400 demonstrate ways in which the presentembodiments are able to provide not only quantitative data (e.g., numberor percentage of cells affected) but also kinetic data (e.g., patternedresponse of the cells over time) and dose-response data (as the amountof test compound contacting cells decreases with distance from the pointof origin of diffusion). By observing patterns of cell responsesoccurring at different distances from the source, the presentembodiments also remove doubt as to whether an actual response (or“hit,” in the language of High Content Screening and High ThroughputScreening assays) is detected, because the responses can be detectedprogressively in accordance with their distance from, and thus theirtime of first contact with, the source of the test compound. Thus, thepresent embodiments may achieve coefficients of variation significantlylower than those of the prior art.

Referring again to FIGS. 3.1-3.2 and FIGS. 4.1-4.4, the small, taperedcentral through-holes 304, 404 of cell activity assay apparatus 300, 400enable a variation on the above-described methods of introducing fluidsinto sites of the apparatus. If the central tapered through-hole 304,404 is used to introduce the cell suspension media (with or withoutcells), and if a pipette is employed for this purpose with a tip-enddiameter larger than the bottom diameter of tapered through-hole 304,404, and the pipette tip is pushed into the tapered through-hole 304,404 until a hydraulic seal is created, then the expression of the cellsuspension introduces the cell suspension media into the cavity 308, 408by hydraulic force. In some assays, this may be advantageous since thecavity 308, 408 is force-filled from the center and cells in the cellsuspension are distributed uniformly within the cavity 308, 408. If thevolume of the expressed cell suspension is slightly larger than thevolume of the cavity 308, 408, the volume of the expressed cellsuspension will provide enough fluid to fill the site out to thehydrophobic barrier 305, 311, 405, 411, (whether that barrier ismechanical, chemical, or both) including the portions of surface 309,409 that form the bottoms of through-wells 302, 303, 402, 403.Alternatively, the volume of the cell suspension media introduced inthis way can be larger than the minimum volume required to fill thecavity 308, 408, and the thin space out to the hydrophobic barrier 305,405, in which case the cell suspension media may partially fill thewells 302, 303, 402, 403. After the cell suspension media has gelled,test compound solution (or tissue 442 as in FIG. 4.2) that is introducedinto a through-well 302, 303, 402, 403 will be farther from the opening,or gap, of the side source 312, 412 into the cavity 308, 408 into whichit will eventually diffuse and set up a concentration gradient (asrepresented by dashed lines 316, 317, 318, 319 (FIGS. 3.2) and 416, 417,418, 419 (FIGS. 4.1 and 4.2)) across the field of cells (as representedby cells 321, 322, 323, 324, 325 (FIGS. 3.2) and 421, 422, 423, 424, 425(FIGS. 4.1 and 4.2)) in the cavity 308, 408. This will cause a delay inthe development of the diffusion gradient in cavity 308, 408, which maybe advantageous for some assays. Alternatively, media with liquid-phasegel, but without cells, may be added to the wells and/or through-hole(302, 303, 304; 402, 403, 404; or some combination thereof) after theinitial introduction of the liquid phase of the cell suspension and itssubsequent gelling. The volume (amount) of this second liquid-phasemedia introduction may determine how high it fills the well and/orthrough-hole (302, 303, 304; 402, 403, 404; or a combination thereof),and consequently how long the delay before the test compoundconcentration gradient forms in the cavity 308, 408.

The embodiments illustrated in FIGS. 3.1-3.2 and FIGS. 4.1-4.4 may alsobe used for more complex assays involving several different compoundsolutions, one diffusing from well 302, 402, another from well 303, 403,and a third from the small center through-hole 304, 404. These methodsmay also be used to ascertain whether and to what extent a test compoundcan inhibit a certain cell activity. For example, a knownchemoattractant can be introduced through the small center through-hole304, 404 (or to a side well 302, 303; 402, 403), and compounds to betested for their ability to inhibit chemotaxis or other cell activitymay be introduced to the other wells. The resulting interactions maythen be compared to a second site in which media with no test compound(or fewer test compounds) is used.

As described above, the present embodiments may allow the determinationof cell activity by detecting changes in cells that are indicative of aresponse to a test compound solution and that occur well before a cellcould migrate through a membrane. These changes include, for example,cell orientation, internal morphological changes, temperaturevariations, molecular movement within the cell, and electromagneticchanges. The most appropriate method of detecting these changes dependsupon the types of compounds and cells that are under investigation. Withany of these methods, the present embodiments may provide the ability tounambiguously detect the kinetics of cell change, based on the fact thatthe test compound concentration gradients, and the cells' activity,advance or progress linearly or concentrically from an opening into thecell suspension.

As one example of detecting cell changes, an infrared reader could beused to monitor changes in the temperatures of cells. The cells could beimmune cells, for example, that are exposed to compounds in the testcompound solution that trigger a metabolic response in the immune cells.This metabolic reaction raises the temperature of the cells. Repeatedscans by the infrared reader detect this rise in temperature. Inaddition, when side and point diffusion sources are used, the infraredreader can observe the effects caused by the concentration gradient, ascells closer to the test compound solution diffusion source respondfirst. The rise in temperature could continue as well, creating atemperature gradient among the cells, which could be another source ofkinetic data.

As another example of detecting cell changes, a light reader, detector,or image-acquisition instrument could be used to monitor movement withinfluorescent-labeled cells. The cells could, for example, be tagged withgreen fluorescent protein. In this manner, well before a cell couldorient itself, change in shape, or move toward the source of the testcompound solution, the reader could document movement internal to thecell.

As another example of detecting cell changes, a microscopic detectionsystem could be used to detect changes in cell shape and orientation. Asa precursor to moving, cells typically undergo morphological changes(e.g., changing their aspect ratio) and orient toward the diffusionsource of a test compound solution. Thus, these changes can be observedwell before the cell migrates. Examples of suitable microscopicdetection systems are the confocal microscopy detection and imagingsystems produced by Atto Bioscience of Rockville, Md.

In another alternative embodiment of the cell activity assay apparatus300 and 400, the test compound solution may be contained in a sinteredmaterial that holds the test compound solution and releases it slowly ina controlled manner.

In the embodiment illustrated in FIGS. 4.3 and 4.4, the site may beprepared by filling cavity 408 with a gelling suspension mediacontaining no cells, which may then be allowed to gel. Then, cellsuspension 420 containing cells may be introduced into through-wells 402and 403, providing a boundary 412 at which the cell suspension 420 is incontact with the gelled suspension media in the cavity 408. The cells433 in through-well 402 may then be treated with a test compound thatmay inhibit cell response. A known chemoattractant 427 may be introducedinto center through-hole 404 and allowed to diffuse concentrically fromcenter through-hole 404 through cavity 408, forming the concentrationgradient represented by lines 435-441 in FIGS. 4.3 and 4.4. Theconcentration would be high at 435, low at 441. The cells in well 403,which have not been treated with the response-inhibiting test compound,respond to the test compound (413-415 in FIG. 4.4), elongating andmigrating toward increasingly concentrated levels of the chemoattractant427, as shown in FIGS. 4.3 and 4.4. Cells 433 in well 402 have beentreated with the response-inhibiting test compound, and are thereforenot responding to the chemoattractant 427, demonstrating that the testcompound is an effective inhibiter.

FIGS. 4.5 and 4.6 are schematic diagrams of one site of a modifiedversion of the assay apparatus of FIG. 4.1, according to an embodiment.In the modified version shown in FIGS. 4.5 and 4.6, the apparatus issimilar to that shown in FIGS. 4.3 and 4.4, except that the recessedarea 413 in the bottom of top plate 401 is deeper than that shown inFIG. 4.3, which results in a larger volume cavity 408 with a greaterdepth between surfaces 413 and 409. The recessed portion 413 of the topplate may be confined to an oval-shaped area that may intersect with thebottom edges of wells 402 and 403 over a limited segment rather thanencircling the wells completely, as in FIG. 4.4. Then, the un-recessedarea 410 of the top plate may have the shape of an oval defined byhydrophobic barrier 405. This oval 405 encloses another oval (area 413)with a circle at each end (through-wells 402 and 403).

FIGS. 4.5 and 4.6 show cells 475 in the cavity 408 and a piece of tumoror other tissue 460, in media, in well 402. As yet no concentrationgradient of compounds emanating from the tissue has formed across thecavity. A cell suspension with cells 475 may have been introduced intothe cavity 408 in ungelled form through tapered through-hole 404, usinga volume of said suspension just sufficient to fill cavity 408,excluding the parts of surface 409 that form the bottoms of wells 402and 403, after which the cell suspension may have been allowed orinduced to gel. With the site thus prepared, a piece of tumor or othertissue 460 in media 488 may be introduced into well 402 (or 403, sincethe wells are symmetrical). Media 488 may be cell-culture media inliquid form. Other cell-culture media 477 and 478 may be introduced intowell 403 and the top portion of the well of through-hole 404 to sustainthe cells in assays with long incubation times. The apparatus may beincubated and then read with an appropriate detection system. Digitalimage HCS detection instruments with image-analysis software may beoptimal for this purpose. The cells 475 may, for example, be killerT-cells that have received a treatment, being tested, that may sensitizethem to compounds emanating from the tissue so that the cells 475 movetoward the tissue. The cells may not respond to compounds diffusing fromthe tissue sample (indicating that the treatment has not beenefficacious), or they may respond by approaching the tissue (indicatingthat the treatment has been effective). Since the deeper cavity 408 maypermit a relatively thick multilayer of cells 475, this embodiment maybe used in studies that must have such a multilayer, for example,longer-term studies of angiogenesis—of the power of tumor tissue toinduce angiogenesis and the ability of test compounds to impede orreverse angiogenesis, among others.

FIG. 4.7 is a schematic diagram of a cross-section of one site of theassay apparatus of FIG. 4.1, having two through-wells 402 and 403, amiddle well with a small tapered through-hole 404, a recessed cavity408, and a mechanical hydrophobic barrier 405. A piece of tumor or othertissue 430 is shown in the middle well, cell monolayers 433 and 434 areschematically depicted in wells 402 and 403, and cell suspensions 428and 429 are shown above the cell monolayers in wells 402 and 403,according to an embodiment. In one method for using the apparatusdepicted in FIG. 4.7, ungelled cell culture media without cells 426 maybe introduced into the apparatus through the center well withthrough-hole 404 in sufficient volume to fill the cavity 408 andpartially fill the wells 402 and 403. The media 426 may be allowed orinduced to gel, and then cell suspensions with cells may be added towells 402 and 403 to form the cell monolayers 433 and 434. The apparatusmay then be incubated until cell monolayers 433 and 434 form.Subsequently, cell suspensions 428 and 429 with cell populations 431 and432 may be introduced into wells 402 and 403, and a tissue sample 430 inmedia 427 is introduced into the center well, with through-hole 404. Theapparatus may then be incubated and then read with a HCS instrument,once or many times, as appropriate, to determine whether and to whatextent the cells in population 431 and 432 (which may be the same ordifferent) respond to the compounds diffusing from the tissue 430 andpenetrating the cell monolayers 433 and 434. Digital image HCS detectioninstruments with image-analysis software are optimal for this purpose.Researchers practiced at the art will appreciate that many other assaysmay be performed with this embodiment.

FIGS. 4.8 and 4.9 are semi-transparent isometric views of one site ofthe side-source multi-site assay apparatus 400 of FIGS. 4.1-4.4 and 4.7.FIG. 4.8 is a schematic 3/4 view of a single site, and FIG. 4.9 is across-sectional view taken along line 4.9 of FIG. 4.8.

FIG. 5 is a top-view schematic diagram of an embodiment of a singlefive-well site of a multi-site assay apparatus 500 having a top plate501 with four through-wells 502, 552, 503, and 553, one in each corner,a central well with a small tapered through-hole 504, a transparentbottom plate 507, and a mechanical or geometric hydrophobic barrier 505enclosing a cavity 508 between the top and bottom plates according to anembodiment. The assay method is similar to above described embodimentsexcept that more options are provided for more elaborate assays, forexample, ones involving four different cell populations deposited intothe four corner wells, and tumor or other tissue in the central well.Alternatively, ungelled cell suspension may be deposited in the cavity508 through the through-hole 504 of the central well, and allowed togel. Various test compounds and or inhibitors then may be deposited inthe corner wells.

FIGS. 6.1-6.3 illustrate top and partial cross-sectional views of anembodiment of an assay apparatus having eight sites, each of which is amulti-well site having a top plate 601 with eighteen peripheralthrough-wells 661-669 and 671-679, two end wells 660 and 670, a taperedcentral through-hole 604, a transparent bottom plate 607, a mechanicalor geometric hydrophobic barrier 605 enclosing the site, and a cavity608 between the top and bottom plates, according to an embodiment. Thisembodiment may be used for long-term cell activity assays in which cellsmay be cultured in the central cavity 608 between the top plate 601 andthe transparent bottom plate 607 of each of the eight sites. In eachsite, ungelled cell suspension may be introduced through the centraltapered through-hole 604 and then allowed or induced to gel. The twentyperipheral through-wells 660-679 may then be filled with standard(non-gelling) cell culture media (which can be replaced periodicallywith fresh media to promote cell growth, cell differentiation, cellproliferation, and tissue development), and the apparatus may beincubated. Periodic or continuous readings of the cell cultures in theeight sites using a HCS instrument or an inverted microscope, may thenbe done. When the cell cultures develop to the desired state (forexample, angiogenesis, development of other kinds of tissue, ordifferentiation of stem cells), test compounds may be introduced throughone or more of the twenty peripheral through-wells to assess theireffect on the cell cultures. Alternatively, one or more cell suspensionsmay be introduced through one or more of the peripheral through-wellsand the interaction may be monitored between these new cells and thecell cultures already in the apparatus. The depth of the cavities (thedistance between the top surface 680 of the bottom plate 607 and thebottom surface 681 of the recessed areas of the top plate 601) can beadjusted to suit the intended assay; if development of thick tissue isdesired, the recessed areas in the top plate 601 may be made deeper.Although for descriptive purposes the embodiment of FIGS. 6.1-6.3includes a particular number of sites, a particular number ofthrough-wells at each site, and a particular layout of the sites andthrough-wells, other embodiments could use different numbers of sitesand through-wells and different layouts, as appropriate for particularimplementations and for desired manufacturing methods.

Although, for clarity, the figures do not show a lid on a cell activityassay apparatus, any of the embodiments described herein could include alid over the apparatus (e.g., over the top plate 101 of FIG. 1.1). A lidmay help minimize evaporation and protect the contents of an apparatusduring transport.

Although embodiments described above illustrate circular through-wellsthat are generally more susceptible to manufacturing (e.g., by drillingor molding), such as through-well 102 in FIG. 1.4, other embodiments mayuse differently shaped through-wells. For example, through-wells may besquare, rectangular, or triangular, which may provide a true linear sideaperture (when viewed from a plan view as in FIG. 1.4) from which todiffuse a test compound. Accordingly, notwithstanding the benefitsassociated with a circular through-well, the present embodiments shouldbe considered broadly applicable to any shaped through-well thatprovides a desired side-source diffusion.

Although embodiments described above involve cell suspensions that maybe incubated to allow cells to settle and adhere to the top surface of abottom plate (e.g., top surface 109 of bottom plate 107 of apparatus 100of FIG. 1.1), alternative embodiments may accommodate assays with cellsthat do not adhere to a surface and instead stay suspended in the cellsuspension media or gelled cell suspension media. These non-adherentcells could include, for example, sperm or bacteria. In these cases, theeffect of the diffusion gradient may still be observed. In other words,even if the cells are moving, a researcher may observe the progressiveeffect as the test compound solution diffuses farther from the sidesource or point source, and reaches and activates additional cells.Where a gel in liquid phase is employed in the cell suspension, andwhere the cell suspension is allowed to gel as in embodiments of thecell activity assay apparatus 200, 300, and 400, the use of non-adherentcell types may be facilitated.

According to other embodiments, a test compound from a compound librarymay be applied in liquid form to a particle or bead. The liquid testcompound may then be gelled or dried on the particle or bead or allowedto be absorbed into the particle or bead. The particle or bead may thenbe submersed in a cell suspension media having cells (either settled andadhered to a surface, or non-adherent), after which diffusion from theparticle into the cell suspension media may occur and the effect on thecells may periodically read. The particle or bead may be coded (e.g.,color coded beads having bands of color) to indicate what testingcompound solution has been applied. The particles or beads may be coatedwith media-soluble coating (e.g., water-soluble). The particles or beadsmay be small vessels, e.g., pieces of capillary tubing or hollow ballshaving a small hole in their surface. In the case of hollow balls, theballs may be filled by applying a vacuum while the balls are in a testcompound solution. When the vacuum is released, the solution may fillthe balls. The particles or beads may also be porous sintered material,e.g., metal, plastic material, glass, or ceramic particles coded bycolor, shape, or some combination thereof. These particles may be filledwith test compound solution using a vacuum in a manner similar to thehollow balls.

The present embodiments are adaptable to a variety of assay formats. Asone example, present embodiments may be applied to assay apparatuscomplying with the Society for Biomolecular Screening (SBS) MicroplateFormat. Such apparatus may employ individual microplates having 8, 16,32, 48, 96, 192, 384, 768, or 1536 sites, which are typically used inautomated systems that assay a high number of compounds. The variousembodiments of the individual site constructions described above may bereplicated across the multiple sites of the SBS plate. However, the useof these individual site constructions is not limited to the SBS format,and may indeed be applied to other standard and non-standard formats.

The present embodiments may provide many surprising benefits, includingbut not limited to one or more of the following:

1) use few cells;

2) use small amounts of test compound;

3) complete assays up to 10-1000 times faster than conventionallive-cell based assays;

4) lower costs by enabling high density studies (i.e., low cost per testper compound);

5) lower coefficients of variation, for example, preventing coefficientsof variation due to pipetting errors from passing through to thecoefficients of variation of the assay, which may enable cell based HCSand HTS;

6) obtain kinetic data as well as quantitative data, in contrast to, forexample, migration assays that count the number or percentage of cellsthat have passed through a filter, but yield no data about the cellsthat have not passed through the filter;

7) reduce reagent costs by virtue of the low number of cells and lowvolume of compounds needed;

8) provide optically advantageous assay apparatus that enable relativelydistortion-free viewing by high-resolution optical detection systems;

9) enable the use of primary cells, e.g., cells directly from patients,as opposed to immortal cell lines;

10) provide apparatus that are geometrically compatible with highresolution optical detection systems, which require flat, transparent,relatively thin bottom plates (between 0.1-2.0 mm thick); and

11) help avoid evaporation problems by confining the cell suspension andtest compounds in an easily-sealed environment.

The benefits of the present embodiments are even more apparent whenconsidering differences from conventional approaches, such as Zigmondchambers. First, adherent cells and only adherent cells are used in aconventional Zigmond chamber. Second, the process of the assay isdifferent. With a conventional Zigmond chamber, the cells are firstplaced on a bottom surface of a top plate (e.g., a cover glass), allowedto adhere to the bottom surface, and then the cells and fluid that arenot in a strip down the middle of the bottom surface of the top plateare completely removed so the top plate is dry except in that middlestrip. The top plate is then inverted onto the middle of the bottomplate (e.g., a 2 mm thick microscope slide with two grooves ground in it4 mm wide and 1 mm deep, with a 1 mm wide ridge in between). The cellsand accompanying media are held between the top plate (e.g., a coverglass) and the top of the ridge of the bottom plate. The top and bottomplate are then clamped together. Then media is wicked into onebottom-plate groove and media with test compound is wicked into theother. The apparatus is then incubated again for a period long enoughfor the cells to respond. The cells that have responded (e.g., elongatedin the direction of the test compound groove) are counted along with thecells that have not responded. If a significant proportion of the totalcell population has responded, it is inferred that the test compound isa chemotactic factor.

The present embodiments (e.g., cell activity assay apparatus 200, 300,400, 500) are distinguishable from conventional approaches, such asZigmond chambers, in many other respects, including but not limited to:

(1) the present embodiments may provide multi-site apparatus;

(2) the present embodiments may enclose cell suspension and testcompound solutions between plates that can be covered or enclosed toprevent or restrict evaporation;

(3) the present embodiments may provide ample volumetric capacity toallow the option of sustaining long term tissue cultures;

(4) in the present embodiments, the volumes of tissue culture media maybe repeatedly renewed without disturbing the cells in the apparatus;

(5) the present embodiments may permit a host of complex assaysinvolving multiple cell types and multiple compounds (see, e.g., cellactivity assay apparatus 200, 300, 400, and 500);

(6) the present embodiments may allow for assays that involve pointsource diffusion, side source diffusion, and both together (see, e.g.,cell activity assay apparatus 300, 400, and 500);

(7) the present embodiments may allow the use of pieces of tissue assources of compounds to effect change in the cells of an assay (see,e.g., cell activity assay apparatus 200, 300, 400, and 500);

(8) the present embodiments may be constructed to alter the spacebetween plates and therefore volume of the cell suspension, whethergelled or otherwise;

(9) the present embodiments may be completely assembled prior to theintroduction of the cell suspension(s);

(10) in configurations as SBS-compatible multi-site assay platforms, thepresent embodiments may enable automated data collection and analysis inhigh content screening platforms (instruments), and the repeatedcollection of data from sites of interest, if any;

(11) the present embodiments may enable the use of non-adherent cellsand adherent cells in the same assay; and

(12) the present embodiments may enable the long term culture of cellsso that the cells may form interconnected tissue, for example, capillarytissue formed from endothelial cells, which tissue may then be studiedfor interaction with other types of cells and compounds or other tissueof interest (see, e.g., cell activity assay apparatus 400 and 500).

Overall, the present embodiments may allow researchers to gather celldata related to, for example, cell orientation, shape, morphology, andintra cellular movements of molecules. In complicated assays such asthose involving angiogenesis, the present embodiments may facilitate thecollection of data on whether angiogenesis is observed. In addition,more data may then be gathered by, for example, adding differentcompounds to the cells undergoing angiogenesis to determine if theangiogenesis can be reversed or inhibited.

Obtaining kinetic data may also significantly lower the coefficient ofvariation of a study. With traditional cell activity assays, largevariations in test sites as well as positive and negative control sitescan result in unacceptably high coefficients of variation. Indeed,researchers usually use duplicate or triplicate sites for each testcompound and repeat traditional cell activity assays two and three timesto confirm results. In contrast, the present embodiments may facilitatekinetic studies that significantly lower the coefficient of variation.

These kinetic studies may identify changes in the cells and cellpopulations progressively. A researcher (or detection system) mayobserve a pattern as cell activity progresses geometrically through apopulation of cells as a test compound diffuses through that population,or observe no progressive pattern. Changes in cell activity progressinggeometrically (in a linear or concentric pattern) in lock step with thediffusion of a test compound in that same pattern may be qualitativelydifferent from assays that cannot use kinetic patterns. Detecting thesepatterns may establish a causal relationship. Therefore, the presentembodiments may virtually eliminate false positives and theirregularities common with current cell based assays.

A researcher using the present embodiments may also determine when cellsthat are responsive to the test compound solution are affecting adjacentcells. For example, by monitoring the diffusion rate of the testcompound solution, a researcher may determine when cells should respond,based on their distance from the side source or point source. If suchcells show changes before that time, then the researcher may deduce thatthey are being affected by adjacent cells before the test compoundsolution reaches them. Such information could be especially valuable forsecondary and tertiary screens.

Although the present description and figures may refer to a single sidesource or a single point source, embodiments may include multiple sidesources or point sources in a site of a cell activity assay apparatus.What is relevant with respect to the number of the diffusion sources isthat they are far enough apart for the kinetics of the phenomena beingstudied to be clear. For example, if there is a pattern of diffusionsources in an assay site, they are preferably far enough apart not tointerfere with one another during the course of the assay. Furthermore,several diffusion sources may be used, one with a known inhibitor of acellular activity and another with a compound from a compound library.As the diffusion front of the inhibitor intersects with the testcompound diffusion front, the pattern of cell activity may show acharacteristic pattern.

The foregoing disclosure of the preferred embodiments has been presentedfor purposes of illustration and description. It is not intended to beexhaustive or to limit the embodiments to the precise forms disclosed.Many variations and modifications of the embodiments described hereinwill be apparent to one of ordinary skill in the art in light of theabove disclosure.

While various embodiments have been described, the description isintended to be exemplary, rather than limiting and it will be apparentto those of ordinary skill in the art that many more embodiments andimplementations are possible that are within the scope of theembodiments. Any feature of any embodiment may be used in combinationwith or substituted for any other feature or element in any otherembodiment unless specifically restricted. Accordingly, the embodimentsare not to be restricted except in light of the attached claims andtheir equivalents. Also, various modifications and changes may be madewithin the scope of the attached claims.

Further, in describing representative embodiments, the specification mayhave presented a method and/or process as a particular sequence ofsteps. However, to the extent that the method or process does not relyon the particular order of steps set forth herein, the method or processshould not be limited to the particular sequence of steps described. Asone of ordinary skill in the art would appreciate, other sequences ofsteps may be possible. Therefore, the particular order of the steps setforth in the specification should not be construed as limitations on theclaims. In addition, the claims directed to the method and/or processshould not be limited to the performance of their steps in the orderwritten, and one skilled in the art can readily appreciate that thesequences may be varied and still remain within the spirit and scope ofthe present embodiments.

What is claimed is:
 1. A multi-well site assay apparatus comprising: afirst plate having an inner surface and an outer surface opposite to theinner surface; a second plate having a first surface and a secondsurface opposite to the first surface, wherein the second surface of thesecond plate faces the inner surface of the first plate; and ahydrophobic barrier on the first plate and/or the second plate, whereinthe hydrophobic barrier defines a test site when the apparatus is viewedfrom a plan view perpendicular to a plane defined by the first plate,wherein the hydrophobic barrier allows passage of gas outside of thetest site while limiting passage of liquid outside of the test site,wherein, within the test site, the second plate defines a first endwell, a second end well, and a through-hole disposed between the firstend well and the second end well, wherein, within the test site, thesecond plate defines a first wall protrusion protruding toward the innersurface of the first plate and extending from the first end well to thesecond end well on a first side of the through-hole, and a second wallprotrusion protruding toward the inner surface of the first plate andextending from the first end well to the second end well on a secondside of the through-hole opposite to the first side of the through-hole,wherein the first wall protrusion and the second wall protrusion definea recess extending from the first end well to the second end well andaround the through-hole, wherein the recess and the inner surface of thefirst plate define a cavity, and wherein the first end well, the secondend well, and the through-hole are in fluid communication with thecavity.
 2. The apparatus of claim 1, wherein the second plate defines aplurality of peripheral through-wells disposed between the hydrophobicbarrier and the recess, when viewed from the plan view.
 3. The apparatusof claim 1, wherein the first wall protrusion and the second wallprotrusion are spaced apart from each other at the first end well and atthe second end well.
 4. The apparatus of claim 3, wherein at the firstend well, between the first wall protrusion and the second wallprotrusion, an inner side of the first end well meets the second surfaceof the second plate at approximately a right angle, and wherein at thesecond end well, between the first wall protrusion and the second wallprotrusion, an inner side of the second end well meets the secondsurface of the second plate at approximately a right angle.
 5. Theapparatus of claim 1, wherein the through-hole comprises a taperedthrough-hole having a wider dimension at the first surface of the secondplate and a smaller dimension at the second surface of the second plate.6. The apparatus of claim 1, wherein the through-hole is positionedgenerally central to the test site when viewed from the plan view. 7.The apparatus of claim 1, wherein the through-hole is positionedgenerally central to the recess when viewed from the plan view.
 8. Theapparatus of claim 1, wherein when viewed from the plan view, the testsite and the recess are elongated in a longitudinal direction runningfrom the first end well to the second end well.
 9. The apparatus ofclaim 8, wherein the first end well, the second end well, and thethrough-hole are generally aligned on a longitudinal line that extendsin the longitudinal direction and bisects the recess.
 10. The apparatusof claim 1, wherein the hydrophobic barrier comprises a groove in theinner surface of the first plate and/or the second surface of the secondplate.
 11. The apparatus of claim 1, wherein the hydrophobic barriercomprises a chemical hydrophobic barrier applied to the inner surface ofthe first plate and/or the second surface of the second plate.
 12. Theapparatus of claim 1, wherein the test site is a first test site, andwherein the first plate and the second plate define a plurality of testsites each substantially identical to the first test site.
 13. Theapparatus of claim 1, wherein the first plate is transparent.
 14. A cellactivity assay method comprising: depositing a cell media containingcells into a cavity of an apparatus, the cavity defined between a firstplate and a second plate of the apparatus, wherein the first plate hasan inner surface and an outer surface opposite to the inner surface,wherein the second plate has a first surface and a second surfaceopposite to the first surface, wherein the second surface faces theinner surface of the first plate, wherein a hydrophobic barrier on thefirst plate and/or the second plate defines a test site when theapparatus is viewed from a plan view perpendicular to a plane defined bythe first plate, wherein the hydrophobic barrier allows passage of gasoutside of the test site while limiting passage of liquid outside of thetest site, wherein, within the test site, the second plate defines afirst end well, a second end well, and a through-hole disposed betweenthe first end well and the second end well, wherein, within the testsite, the second plate defines a first wall protrusion protruding towardthe inner surface of the first plate and extending from the first endwell to the second end well on a first side of the through-hole, and asecond wall protrusion protruding toward the inner surface of the firstplate and extending from the first end well to the second end well on asecond side of the through-hole opposite to the first side of thethrough-hole, wherein the first wall protrusion and the second wallprotrusion define a recess extending from the first end well to thesecond end well and around the through-hole, wherein the recess and theinner surface of the first plate define a cavity, and wherein the firstend well, the second end well, and the through-hole are in fluidcommunication with the cavity; allowing the cell media to wick into thecavity to the first wall protrusion and the second wall protrusion;placing a test material in the first end well and in contact with thecell media; diffusing into the cell media the test material, wherein afirst cell of the cell media is closer to the first end well than asecond cell of the cell media; allowing the test material to diffuseinto the cell media for a predetermined duration such that the firstcell is exposed to the test material longer than the second cell isexposed to the test material; after the predetermined duration,detecting a degree of activity of the first cell and a degree ofactivity of the second cell; and comparing the degree of activity of thefirst cell to the degree of activity of the second cell to determine thepresence or absence of a progressive cell activity response by the cellsof the cell media to determine whether the test material inducesprogressive cell activity response.
 15. The method of claim 14, whereindepositing the cell media into the cavity comprises depositing anungelled cell suspension through the through-hole and then allowingand/or inducing the ungelled cell suspension to gel.
 16. The method ofclaim 14, wherein before placing the test material, the method furthercomprises: depositing a cell culture media in the first end well and/orthe second end well; incubating the apparatus; and reading cell cultureof cells of the cell media.
 17. The method of claim 14, wherein the testmaterial comprises a first test material, wherein the second platedefines a plurality of peripheral through-wells disposed between thehydrophobic barrier and the recess, when viewed from the plan view, andwherein the method further comprises: placing a second test material ina first peripheral through-well of the plurality of peripheralthrough-wells and in contact with the cell media, wherein the secondtest material is different from the first test material, diffusing intothe cell media the second test material, wherein a third cell of thecell media is closer to the first peripheral through-well than a fourthcell of the cell media; allowing the second test material to diffuseinto the cell media for a second predetermined duration such that thethird cell is exposed to the second test material longer than the fourthcell is exposed to the second test material; after the secondpredetermined duration, detecting a degree of activity of the third celland a degree of activity of the fourth cell; and comparing the degree ofactivity of the third cell to the degree of activity of the fourth cellto determine the presence or absence of a progressive cell activityresponse by the cells of the cell media to determine whether the secondtest material induces progressive cell activity response.
 18. The methodof claim 14, wherein the test material comprises a test compoundsolution or a cell suspension.
 19. A multi-well site assay apparatuscomprising: a first plate having an inner surface and an outer surfaceopposite to the inner surface; a second plate facing the first platesuch that a cavity is defined between the second plate and the firstplate, wherein the second plate has a first surface and a second surfaceopposite to the first surface, the second surface facing the innersurface of the first plate, wherein the second plate defines a first endwell and a second end well, each extending from the first surface of thesecond plate to the second surface of the second plate and in fluidcommunication with the cavity; and a hydrophobic barrier on the firstplate and/or the second plate, wherein the hydrophobic barrier defines atest site when the apparatus is viewed from a plan view perpendicular toa plane defined by the first plate, wherein the hydrophobic barrierallows passage of gas outside of the test site while limiting passage ofliquid outside of the test site, wherein the first end well is disposedat a first side portion of the test site when viewed from the plan view,and the second end well is disposed at a second side portion of the testsite opposite to the first side portion, wherein, when viewed from theplan view, the test site has a length from the first side portion to thesecond side portion, and the length is greater than a width of the testsite, wherein the first plate and the second plate define a first gapbetween the first plate and an edge of the first end well at the secondsurface of the second plate, on a side of the first end well facing thesecond side portion of the test site, and wherein the first plate andthe second plate define a second gap between the first plate and an edgeof the second end well at the second surface of the second plate, on aside of the second end well facing the first side portion of the testsite.
 20. The apparatus of claim 19, wherein, within the test site, thesecond plate defines a first wall protrusion protruding toward the innersurface of the first plate and extending from the first gap of the firstend well to the second gap of the second end well on a first lateralside of the test site, and a second wall protrusion protruding towardthe inner surface of the first plate and extending from the first gap ofthe first end well to the second gap of the second end well on a secondlateral side of test site opposite to the first lateral side of the testsite, wherein the first wall protrusion and the second wall protrusiondefine a recess extending from the first end well to the second endwell, wherein the recess, the inner surface of the first plate, thefirst wall protrusion, and the second wall protrusion define the cavity,and wherein the second plate defines a plurality of peripheralthrough-wells that are disposed between the hydrophobic barrier and therecess when viewed from the plan view, and are in fluid communicationwith the cavity.